In-vitro mechanical loading of musculoskeletal tissues

ABSTRACT

Musculoskeletal tissues produced in vitro are optimized in response to an externally applied mechanical load. The load applied may vary from tissue to tissue, depending upon the response desired, and may include intermittent axial, torsional, and bending loads to produce cortical structures. Compression alone is preferably applied to produce cancellous bone. A method according to the invention for culturing bone in vitro comprises: providing a culture vessel providing a scaffold material, supporting the scaffold material within the tissue culture vessel so as to be exposed to a tissue culture medium, and exerting a force on the scaffold material during growth of a bone construct (a cultured bone growth) around the scaffold material. Applicable apparatus preferably includes a culture vessel, holders for holding a scaffold within the tissue culture vessel, means for introducing a tissue culture medium to the tissue culture vessel, and an actuator adapted to apply a force to developing bone during the in vitro culture of the tissue, whether bone, cartilage, ligament, or composites thereof.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.09/850,659, filed May 7, 2001, which claims priority from U.S.provisional patent application Ser. No. 60/202,282 filed May 5, 2000,the entire content of both of which is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the in-vitro growth of musculoskeletal tissues,in particular bone, cartilage, and ligaments.

BACKGROUND OF THE INVENTION

Musculoskeletal tissues are composed of a composite of cellular andmatrix components. In vivo, the cells are generally believed to bederived from undifferentiated cell lines that respond to differentstimuli, both chemical and mechanical, and then ultimately differentiateand produce a particular matrix providing a tissue with a givenstructure and function. Furthermore, musculoskeletal tissues in livingorganisms have the ability to adapt to mechanical and physiologicchanges throughout life.

An example is bone. The material properties of bone are governed by thedensity (and microdensity) of the material. The geometry of the bonedetermines its strength. The tubular structure of long bones providesthem with a greater moment of inertia than would be true if bones weresolid rods. Consequently, bones are stronger withstanding bending ortorsional stresses than they would be if they were solid rods. As onegrows older, the outer diameter increases as does the inner periostealdiameter. Theoretically, these changes allow one to maximize bonestrength as bone mass decreases with age.

Articular cartilage is similarly composed of cellular and matrixcomponents. The cells are uniquely isolated by the matrix and highlyresponsive to their environment. The matrix is composed primarily ofcollagen, proteoglycan, and water. The three-dimensional lattice andhydrostatic forces give cartilage its unique ability to withstandcompressive forces.

In addition to what has been observed in-vivo, in-vitro studies haveshown chondrocytes respond to mechanical loads (P. M. Freeman et al., J.Orth. Res., 12(3), 311-319 (1994)). This study found a decrease in thecell volume of chondrocytes in response to compressive loads. Otherstudies have shown an increase in proteoglycan synthesis and depositionin response to intermittent physiologic compression (G. P. J. van Kampenet al., Arthritis Rheum. 28 419-424 (1985)). Bone changes in response toload have been documented for many years and the appositional depositionof bone in an effort to increase the structural strength of loads areasis generally referred to as “Wolff's Law.” Similarly, tendon andligament healing has been shown to be affected by the forces applied tothese tissues at various periods in the healing process.

The last few years have seen a rapid increase in the number ofbiomaterials available to augment and enhance the body's ability torepair and replace damaged musculoskeletal tissues. A recent article inthe New England Journal of Medicine discusses autologous cartilagetransplantation as a treatment of deep cartilage defects in the knee (M.Brittberg et al., New England J. Medicine, 331(14) 889-895 (1994). Thismethod is currently available in the United States and undergoinginvestigation. The patient's cartilage is essentially “cloned” andreinserted in a cartilaginous defect after being grown in vitro to theappropriate volume. It is injected as a liquid paste and secured by anautologous periosteal patch. The authors had “encouraging” results infemoral condyle defects although the results were poor in the highlymechanically loaded patella.

Bone morphogenic protein, growth hormone, coral bone substitutes, bonepaste, etc. are commercially available products used to enhance repairof fractures, nonunions, or osseous defects. These materials are gainingwidespread acceptance within the medical community for theirapplicability in complex cases. Most of these products lack mechanicalstrength and structural properties approximate to the tissues they willsupport and rely on the healing of the host before adequate function canbe restored

In U.S. Pat. No. 6,121,042, Peterson et al. disclose an apparatus forapplying an axial load to a cultured tendon or ligament construct.However, the growth of bone is not disclosed. Further the application oftorsional forces is not disclosed. Peterson further fails to disclosethe application of forces scaled by (such as proportional to) a relevantelastic modulus of the cultured structure.

SUMMARY OF THE INVENTION

Broadly, this invention optimizes musculoskeletal tissues producedin-vitro by utilizing their unique ability to respond to mechanicalload. The load applied may vary from tissue to tissue, depending uponthe response desired. For example, intermittent axial, torsional, andbending loads can be applied to bone cells and matrix when the desiredresponse is to produce tubular bone. Compression alone is preferablyapplied to produce cancellous bone.

An improved method for culturing bone in vitro comprises: providing aculture vessel providing a scaffold material, supporting the scaffoldmaterial within the tissue culture vessel so as to be exposed to atissue culture medium, and exerting a force on the scaffold materialduring growth of a bone construct (a cultured bone growth) around thescaffold material.

An improved apparatus for culturing bone in vitro comprises: a culturevessel, holders for holding a scaffold within the tissue culture vessel,means for introducing a tissue culture medium to the tissue culturevessel, and an actuator adapted to apply a force to developing boneduring the in vitro culture of the tissue, whether bone, cartilage,ligament, or composites thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a system for in vitro growth of bone according to thepresent invention;

FIG. 2 shows a system embodiment;

FIG. 3 is a flow chart illustrating an improved method of in vitro bonegrowth;

FIG. 4 is a flow chart illustrating a method of applying a forcecorrelated with a measured physical property of the growing bone matrix;

FIG. 5 is a flow chart illustrating a further method of applying a forcecorrelated with a measured physical property of the growing bone matrix;

FIG. 6 shows another system for in vitro growth of bone according to thepresent invention, using a motorized actuator to apply a force;

FIG. 7 shows another system for in vitro growth of bone according to thepresent invention, using magnetic repulsion to apply a force;

FIG. 8 shows another system for in vitro growth of bone according to thepresent invention, in which the force is applied through a flexiblecomponent of the culture vessel enclosure;

FIG. 9 is a drawing of a joint simulator disposed within a culturevessel according to the invention;

FIG. 10A shows the used of force applied directly to a culture vessel;

FIG. 10B also shows the used of force applied directly to a culturevessel; and

FIG. 11 shows an alternative “piston-like” culture vessel according tothe invention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a system applicable to the in-vitro culturing of bone ortissue according to the present invention. Culture vessel 10 has aculture medium inlet conduit 12, having an inlet control 14, and aculture medium outlet conduit 16, having an outlet control 18. Ascaffold material 20 is supported in culture medium 22 by first holder24 and second holder 26.

The culture medium is enclosed by culture vessel 10 and inner lid 28.Inner lid 28 is supported, relative to the walls of vessel 10, by one ormore pins such as 30. A spacer 32 passes through a hole in the inner lid28, maintaining a seal, and connects to actuator 36 and force sensor 34to outer lid 38, held in place relative to vessel 10 by one or more pins40. Electrical connections 42 and 44 lead to electrical contacts 46 and48, allowing power to be provided to actuator, and signal to be obtainedfrom the force sensor.

Scaffold Material

The scaffold material can be any porous, fibrous, meshed, woven, orother material suitable for growth of bone material. In a preferredembodiment, the scaffold is formed from collagen fibers. In alternativeembodiments, the scaffold material may be a porous glass, sol-gel,aerogel, xerogel, ormosil, polymer gel, porous ceramic, proteins,biomaterial, hydroxyapatite framework, nylon, other biocompatiblepolymer, or other biocompatible material, including pieces of bone,cartilage, ligament, or other appropriate tissue. The scaffold mayprovide intrinsic structural integrity, or may be a flexible fibernetwork.

Polymer gels may be grown in solvent, then dried for use as a scaffoldmaterial. Gels may be cross-linked by UV, other radiation exposure, orother chemical means before use as a scaffold material. The scaffoldmaterial may be a piece of autogenous tissue (i.e., obtained From apatient), to ensure biocompatibility any reduce the likelihood of ahost/graft rejection.

Culture Medium

The culture medium contains a mixture so as to induce growth of bonematerial on the scaffold. Preferably, this is an aqueous solutioncontaining osteoblasts. Other culture medium components may include:protein sources, carbohydrates, fats, and minerals for depositionincluding calcium and phosphate. Any appropriate enzymes, co-enzymes,hormones or growth modifiers may be included as well. Stem cells canadvantageously be derived from fat or other tissue obtained from thepatient receiving the implant, and used in the culture medium for any ofthe embodiments of the present invention.

Actuator

Actuator 36 is preferably a stacked piezoelectric actuator, so as toprovide stress (compression force) and/or strain (extensive force) tothe scaffold and growing matrix through a displacement of the upper endof the scaffold. Other actuators may be used, such as solenoids orelectric motors. The matrix itself may be bone, cartilage, or ligamenttissue produced by osteoblasts, chondroblasts, and fibroblasts,respectively. Pluripotential stem cells can differentiate into thevarious cell lines, depending upon the environment created by theculture medium and force construct utilized.

The signal from force sensor 34 can be used to control the force appliedto the scaffold, and thereby to the bone matrix on the scaffold. Thecompression modules can be determined for the displacement of theactuator and the force signal provide by the force sensor. Initially,the force signal may be very small or zero, particularly if a fibrousscaffold is used, and compressive force is applied. As a bone matrixdevelops, the force signal will increase as the bone provides resistanceto compression. The displacement applied by the actuator can becorrelated with the force signal. For example, the displacement appliedby the actuator can be controlled to be correlated with the force signalobtained per unit displacement.

System Embodiment

FIG. 2 shows a system according to the present invention. A computer 50receives a force signal from force sensor 52. The computer 50 also has asoftware application program adapted to send a signal to the actuatorcontrol, so as to control the actuator.

Compression Modulus

The program determines the compression modulus of the bone growth fromthe ratio of the force measured to actuator displacement. The actuatoris then controlled so as to apply a force correlated (for example,proportional to) the compression modulus of the bone.

An effective compression modulus M can be defined by

$M = \frac{\Delta \; X}{F}$

where F is the applied force, and ΔX is the actuator displacement orother equivalent measure of bone compression.

Equivalently, the modulus can be determined by applying a force to acultured structure, and monitoring the consequent displacement. A forceprovider and displacement sensor can be used in place of an actuator(providing displacement) and a force sensor.

Improved Method of In Vitro Culture

FIG. 3 shows a flow chart illustrating an improved method of preparing acultured structure. The cultured structure may be bone, cartilage,ligament, skin, tendons, organ components such as heart valves, bloodvessel components, other connective tissue, other cellular tissue,teeth, or other biological materials.

Box 60 corresponds to the provision of a culture vessel, such asdescribed in relation to FIG. 1.

Other culture vessels known by skill of those in the art may beadvantageously adapted for use with the present method, for example,such as described in U.S. Pat. Nos. 5,153,136 to Vanderburgh; and4,839,280; 6,037,141; 6,048,723 to Banes; 6,171,812 to Smith and6,121,042 to Peterson. The entire contents of any patent or publicationreferred to in this specification are incorporated herein by reference.

Box 62 corresponds to the support of a scaffold material within theculture vessel. Suitable scaffold materials have been discussed above.Box 64 corresponds to the exposure of the matrix material to a culturemedium so as to grow the cultured matrix structure on and around thescaffold. The culture vessel will be fully or partially filled withculture medium. In a preferred embodiment, an aqueous solutioncontaining osteoblasts is used. (The scaffold material may also beexposed to a gel containing osteoblasts, or other culture mediumcomponents discussed above). The culture medium can be periodically orcontinuously replaced by fresh culture medium. The culture medium withthe vessel may also be stirred or agitated.

Box 66 corresponds to the monitoring of the cultured structure. Thismonitoring will be discussed in detail below. Box 68 corresponds to theapplication of a force to the cultured matrix structure, so as toimprove the properties of the structure. In the case of bone culture, acompressive force is preferably applied to the culture so as to improvethe development and mechanical properties of the cultured matrixstructure. The apparatus and systems described above are preferablyused. Other embodiments and methods are described below.

Box 70 corresponds to the removal of the culture from the culturevessel. Other processes can be applied to the culture after removal fromthe vessel, and before implantation. These may include cleaning,shaping, further mechanical processing (such as loading), sterilizationand other characterization tests such as ultrasound density measurementsand immunological tests. An unsatisfactorily cultured matrix structuremay be discarded, returned to the culture vessel, or otherwise furtherprocessed before implantation. Box 72 corresponds to implantation of thecultured structure into a patient.

Monitoring of Cultured Structure

During early stages of bone culture, it may be unhelpful to apply forcesto the cultured bone, as the culture may be overly brittle at thisstage. The culture process may be divided into two or more periods; thefirst period with no forces applied, and the later stage(s)characterized by the application of forces.

The bone culture can be visually inspected by a technician during growthto monitor expected development and to initiate corrective measures ifdevelopment is less than expected. The walls of the culture vessel maybe in full or in part transparent, or windows provided, to allow visualmonitoring. Ultrasound, x-rays, and other radiation/tomography may beused to monitor the density of bone during in-vitro culture. The x-rays,MRI and/or CAT scans may be analyzed automatically, through patternrecognition, for example.

Electric fields generated from bone compression may also be monitored.An oscillating compression can be applied to the bone sample and aresulting electric field detected and monitored so as to characterizethe properties of the bone. This may be achieved even in a conductingmedium if the signal frequency is rapid, compared with ion diffusiontime in the conducting medium. The detected electric field is related tothe piezoelectric properties of bone, which are correlated with themechanical strength of the bone. Hence, a force can be applied to thebone culture scaled according to the detected piezoelectric signal.

The force applied to the bone can also be scaled according to anestimate elastic modulus (Young's modulus, compression modulus, andsimilar) based on ultrasonic, x-ray, or other radiation absorptionmeasurements.

Acoustic waves can also be used to characterize the cultured bonesample, e.g. from the noise generated by a mechanical impulse. Theresonant frequencies of a cultured bone sample can be used to determinemechanical strength.

Total applied forces may be limited by known properties of nativebiological material.

Force Application

In preferred embodiments, a compressive force is applied to a culturedbone sample, and the magnitude of the applied force is correlated withthe mechanical and/or structural properties.

It can also be beneficial to apply tension to a cultured structure, suchas a tendon, ligament, or cartilage. In this case, the Young's modulusof the structure can be determined, and a tensioning force appliedhaving a magnitude correlated with (e.g. proportional to) the Young'smodulus.

It can further be beneficial to apply an oscillating force to thecultured structure, having compressive and tensioning components.Conventionally, a symmetrical oscillating force can be applied, howeverby determining elastic moduli for tension and compression separately, anon-symmetrical oscillating force can be applied, having, a tensioningcomponent scaled by the tension modulus; and a compression componentscaled by the compression modulus.

It can further be beneficial to apply a torsional (twisting) force tothe cultured structure during its growth. One end of a cultured bonesample can be fixed, relative to the culture vessel, and a rotatingactuator such as a micro-stepper motor used to apply a twist to theother end of the bone sample. The torsional modulus can be determined,and the angular displacement scaled by the torsional modulus.

In all cases of force application, the appropriate modulus can bedetermined as the force is applied and increased. If the behavior of themeasured modulus indicates a yield point, fracture, or other structuralbreakdown, the force then can be limited to lower values.

A microphone can be used to detect signals characteristic of structuralbreakdown during the application of force(s), and the signals used tolimit the maximum applied force. A maximum force can be defined by knownproperties of fully cultured tissue.

Force Control

FIG. 4 shows a flow chart illustrating a method of controlling a forceapplied to a cultured structure (bone, ligament, cartilage, and thelike) during growth. For convenience, this method concerns applicationof a compressive force, but this is intended to be non-limiting, astension, torsional, bending and combination forces can also be appliedaccording to this method.

Box 80 corresponds to the application of a displacement to the culturedstructure. For example, using a piezoelectric actuator, the displacementis correlated with the voltage applied to the actuator. Using a steppermotor, the displacement is correlated with the number of step pulsesapplied. Actuators can also comprise an integrated displacement sensor.

Box 82 corresponds to the measurement of the force on the structurecorresponding to the applied displacement. A signal from a force sensoris obtained.

Box 84 corresponds to the determination of the modulus of the structure.An electronic device, such as a computer, can be used to control theactuator, receive a signal from the force sensor, and determine amodulus using a software program or algorithm

Box 86 corresponds to the application of a displacement to the structurecorrelated with the modulus. A stronger structure has a stronger forceapplied to it, up to a predetermined maximum force.

FIG. 5 illustrates a method similar to that shown in FIG. 4, except thata known force is applied to the structure, and a correspondingdisplacement is measured.

Box 90 corresponds to the application of a known force, Box 92corresponds to a determination of corresponding compression, extension,twist, bend or other deformation, and Box 94 corresponds to theapplication of a force with a magnitude correlated with the determinedmodulus.

In another embodiment, a compressive force is applied to the end of acultured bone and the consequent bending determined, allowing an elasticmodulus to be determined.

Displacement sensors can use any conventional technique, such asmicrometers, laser reflection, capacitance, and other effects.

Force sensors also can use any conventional technique, such aspiezoelectric effects, and other stress and/or strain sensors.

Other Embodiments

Other means of applying forces to a cultured construct include using apumped fluid (such as compressed air), a flexible or elastic structureor sheet, a thermal expansion element, other piezoelectric or electricalinduced expansion elements, gravity, magnetic fields (such as attractionor repulsion between magnetic elements), shock waves, acoustic waves,impacts, motors, expansion chambers, or other elements or devices whichexpand, contract, or deform in a controllable manner.

FIG. 6 shows a device in cross-section having frame 100, supporting aculture vessel comprising cylindrical housing 102 and housing end 104.Scaffold 108, of some biocompatible material, is supported by first andsecond supports 106 and 110. The frame 100 supports a motor-drivenmicrometer 114, having shaft 112 controlling position of housing end104, and hence the force on a cultured bone grown on the scaffold 108.The motor driven micrometer comprises a force sensor. Micrometerposition and force readings are electrically accessible through contacts116. The housing 102 can be rotated about the long axis periodically toaverage the effects of gravity. Growth culture medium inlet and outletconnectors are provided on the housing 102 or housing end 106 (notshown). The shear flows generated by housing rotation can further beused to improve the properties of grown culture matrix constructs.

FIG. 7 shows another system having a culture vessel 120 furthercomprising an electromagnet 122 embedded in the vessel, theelectromagnet providing a variable repulsive force to suitable alignedmagnet 124, the magnet propagating the force to scaffold 128 (or cultureor matrix) through scaffold attachment 126. Scaffold holder 130 isconnected to force sensor 132, which measures the force applied to thescaffold 128. This system is advantageous over other systems (such asdisclose in U.S. Pat. No. 6,191,042) in that the electromagnet iscontained within the culture vessel, and in that a repulsive magneticforce is applied.

FIG. 8 shows a further culture vessel having housing 150, containingflexible membrane 152. culture medium is contained by the housing belowthe flexible membrane. Scaffold 148 is supported by first and secondsupports 142 and 144, and support 144 is connected to the membrane byspacer element 146. An electrically controlled actuator 154, havingelectrical contacts 156, applies a compressive force to the scaffoldthrough shaft 152 and through the membranes.

The supports or holders for the scaffold material can be any convenientdesign, such as clips, adhesives, cement, hook-and-loop structures(hooks and/or loops can be provided by the matrix), and the like.

Other mechanisms can be used to apply compressive or extensive force tothe scaffold and growing matrix construct through the membrane, such asgas pressure (or vacuum), weights placed on the membrane (which can beincreased with time), expansion chambers, expanding materials such ashydrating gels, springs, torsion wires, elastic structures, compressedresilient materials, and the like. In other embodiments, such mechanismscan be used in place of the actuator shown in the system of FIG. 1.Acoustic waves and mechanical shock waves can also be applied to providetissue compression.

In addition to the above-referenced mechanical constructs, a “jointsimulator” such as that used for testing prosthetic joint implants orperforming experimentation (including cadaveric joints) may be used withappropriate load cells applied to recreate the appropriate forces toinduce a desired matrix construct from cell culture. An applicablefixture is illustrated in FIG. 9 with respect to a knee simulator withina culture vessel, with the understanding that other joints, includingthe hip, shoulder, spine, elbow and those within the hands and feet maybe accommodated through appropriate modification. In FIG. 9, a distalfemur is shown at 902, interacting with a proximal tibia 908. Anactuator 906 is used with respect to the distal femur, in conjunctionwith a force sensor 904, whereas actuator 910 is used with sensor 909relative to the tibia 908. Flow into the vessel through conduit 912 isregulated at 914, with flow out through conduit 916 being regulated bydevice 918. A scaffold vessel for chondrocytes is depicted at 990, alongwith optional flexible fusion 991. Forces are applied through theactuator(s) to simulate the load of walling, compression, gliding andtorsion may be simultaneously simulated as well.

Through the use of a system such as that depicted in FIG. 9, the culturevessel, with appropriate scaffold, positioned as necessary to simulatethe forces encountered in a natural joint. In this way, multiple smalltissue matrix constructs, or fewer larger tissue matrix constructs, maybe developed simultaneously. In addition, composite tissue constructsmay be created, such as bone-tendon-bone or cartilage-bone, which wouldmake implantation into patients more predictable. Either a jointsimulator of the type shown in FIG. 9 or alternative, modified systemsto use with one or more scaffolds with one or more cultured vessels andmultiple cell lines or pluripotential stem cells exposed to differentmedia and forces may be used to illicit the production of compositematrix components according to the invention.

Through the use of certain of the chambers referenced above, it may befurther possible to induce and modify matrix constructs without the needfor a pre-existing scaffold. As shown in FIGS. 10A and 10B, a deformableculture vessel containing culture medium 210 and inlet/outlet ports 214and 212, respectively, may be modified by force 202 through actuator 200to provide a compression of the medium 210, or, with a force of adifferent direction such as 203 shown in FIG. 10A, a tension may berealized. As described elsewhere herein, the force may be long-term,intermittent, or even randomly effectuated, through oscillation, forexample, using any of the actuation devices herein disclosed.

FIG. 11 depicts a further option in the form of a “piston” type ofculture vessel including portions 302 and 304 movable and rotatable withrespect to one another by virtue of a flexible seal. The culture medium310 may be introduced through port 314 and expelled through port 316,while compression, tension, or torsional forces may be applied to eitherside of the system, through oscillatory or other types of interaction.

In addition to, or instead of, mechanical forces, cultured tissue can beexposed to other processes to improve properties. In the case of bonegrowth, the cultured bone may be exposed to electromagnetic radiation,electric fields, magnetic fields, electrolytic effects, chemicalexposure, biomolecule exposure (e.g. exposure to enzymes, hormones, andthe like), thermal processes such as thermal cycling, chemical effectsincluding photochemical effects, ion implantation, other radiationexposure including ultrasound exposure, and other effects so as toimprove bone quality. These processes can be performed on tissuecultures within the culture vessel, or where appropriate, outside of thevessel, such as prior to implantation in a patient.

The matrix structure (or scaffold) can be non-uniform in cross section.For example, the density can be higher around the edges. Using a fibrousscaffold to support bone growth, the fiber density can be higher aroundthe periphery of the matrix structure. This approach is useful to growbones having a natural structure with a higher density around theperiphery. The force applied to the bone construct can also benon-uniform, for example using an array of actuators. For example, usingan array of programmable actuators, such as piezoelectric actuatorscontrolled by a computer system, a higher force can be applied aroundthe periphery of the bone construct, so as to encourage the growth ofstronger bone around the periphery. The force can be lower in centralregions. The force applied to the bone growth can be correlated with thedensity of the bone growth, for example as determined using ultrasonicattenuation. The force applied can also be correlated with thepiezoelectric response of the bone construct. The culture environment ofthe bone growth can also be varied, for example through release ofchemicals, hormones, bioactive agents and the like, through additionalfluid inlets, local heating, time-release elements incorporated into thematrix structure, variable surface processing of the scaffold material,non-uniform irradiation of the growing structure (e.g. withelectromagnetic radiation), and the like. Scaffold materials can also bederived from organic sources, such as animal bone processed tosubstantially remove organic components.

1. A method of synthesizing musculoskeletal tissue, the methodcomprising the steps of: providing a cell culture vessel containing acell culture medium; introducing at least one musculoskeletal precursorto the medium; applying a force to the musculoskeletal precursor invitro through an actuator that mimics forces experienced by a bone in abody, whereby the precursor develops into a musculoskeletal tissuematrix having desired properties through the application of the force.2. The method of claim 1, wherein the culture medium includesosteoblasts.
 3. The method of claim 1, wherein the culture mediumincludes chondroblasts.
 4. The method of claim 1, wherein the culturemedium includes fibroblasts.
 5. The method of claim 1, wherein theculture medium includes stem cells.
 6. The method of claim 1, whereinthe matrix develops into bone tissue.
 7. The method of claim 1, whereinthe matrix develops into cartilage.
 8. The method of claim 1, whereinthe matrix develops into ligament.
 9. The method of claim 1, wherein thematrix develops into tendon.
 10. The method of claim 1, wherein thematrix develops into a bone-tendon-bone composite.
 11. The method ofclaim 1, wherein the matrix develops into a cartilage-bone composite.12. The method of claim 1, wherein the tissue matrix includes stemcells.
 13. The method of claim 1, wherein the actuator that mimicsforces produced in a natural bone joint.
 14. The method of claim 1,wherein the actuator that mimics forces produced in a knee joint. 15.The method of claim 1, wherein the actuator that mimics forces producedin a hip joint.
 16. The method of claim 1, wherein the actuator thatmimics forces produced in a shoulder joint.
 17. The method of claim 1,wherein the actuator that mimics forces produced in an elbow joint. 18.The method of claim 1, wherein the actuator that mimics forces producedin a spine.
 19. The method of claim 1, wherein the actuator applies acompressive force.
 20. The method of claim 1, wherein the actuatorapplies tension.
 21. The method of claim 1, wherein the actuator appliesan oscillating force.
 22. The method of claim 1, wherein the actuatorapplies a torsional or twisting force.
 23. The method of claim 1,wherein intermittent axial, torsional and bending loads are applied toproduce a tubular bone.
 24. The method of claim 1, wherein compressionalone is applied to produce cancellous bone.
 25. The method of claim 1,further including the steps of: supporting a scaffold material with theculture vessel; and applying the force to the scaffold material throughthe culture medium so as to synthesize the tissue on the scaffoldmaterial.
 26. The method of claim 1, further including the step ofmeasuring a physical property of the tissue during the synthesisthereof.
 27. The method of claim 26, wherein the magnitude of the forceis correlated with the measured property.
 28. The method of claim 26,wherein the physical property is an elastic modulus or a Young'smodulus.